Search results for: stem cell differentiation

Authors: Yin-Ching Keung, Kit-Lun Yick

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The issue of bra fitting has persisted throughout history despite advancements in molded bra cups. To gain a deeper understanding of the interaction between the breast and bra pattern, this study combines the art of traditional bra patternmaking with 3D body scanning technology. By employing a 2D bra pattern drafting method and analyzing the effect of body shape on the desired bra cup shape, the study focuses on the differentiation of the lower cup among bras designed for flat and round body-shaped breasts. The results shed light on the impact of body shape on bra fit and provide valuable insights for further research and improvements in bra design, pattern drafting, and fit. The integration of 3D body scanning technology enhances the accuracy and precision of measurements, allowing for a more comprehensive analysis of the unique contours and dimensions of the breast and body. Ultimately, the study aims to provide individuals with different body shapes a more comfortable and well-fitted bra-wearing experience, contributing to the ongoing efforts to alleviate the longstanding problem of bra fitting.

Keywords: breast shapes, bra fitting, 3D body scanning, bra patternmaking

Procedia PDF Downloads 63

Authors: Mehdi Ghanavati

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The Effects of salinity level and duration on carbon isotope discrimination (Δ) of Matricaria chamomilla and Matricaria aurea were evaluated. Four ecotypes of M. chamomilla and four ecotypes of M. aurea were grown at different NaCl concentrations (control, 6, 12 and 18 dS/m) in sand culture condition. Carbon isotope discrimination (Δ) varied significantly (p<0.001) among ecotypes. The amount of carbon isotope discrimination (Δ) increased in first salinity level (6 dS/m), but in other levels (12 and 18 dS/m) it did not increase. Stages of salinity treatments (two stages: first from seedling stage until the end of the experiment and second stage of stress exertion began at stem elongation and seedlings emergence from rosette stage to harvest) had not a significant difference. Study of two spices of chamomile showed the M. aurea had a higher amount of carbon isotope discrimination (Δ) (22.9%) than M. chamomilla (22.48%).

Keywords: salinity, carbon isotope discrimination, Matricaria chamomilla, Matricaria aurea

Procedia PDF Downloads 442

Authors: Sirus Mohammadi, Rohala Moghimi

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The main advantages of microgrids are high energy efficiency through the application of Combined Heat and Power (CHP), high quality and reliability of the delivered electric energy and environmental and economic advantages. This study presents an energy management system (EMS) to optimize the operation of the microgrid (MG). In this paper an Adaptive Modified Firefly Algorithm (AMFA) is presented for optimal operation of a typical MG with renewable energy sources (RESs) accompanied by a back-up Micro-Turbine/Fuel Cell/Battery hybrid power source to level the power mismatch or to store the energy surplus when it’s needed. The problem is formulated as a nonlinear constraint problem to minimize the total operating cost. The management of Energy storage system (ESS), economic load dispatch and operation optimization of distributed generation (DG) are simplified into a single-object optimization problem in the EMS. The proposed algorithm is tested on a typical grid-connected MG including WT/PV/Micro Turbine/Fuel Cell and Energy Storage Devices (ESDs) then its superior performance is compared with those from other evolutionary algorithms such as Genetic Algorithm (GA), Particle Swarm Optimization (PSO), Fuzzy Self Adaptive PSO (FSAPSO), Chaotic Particle PSO (CPSO), Adaptive Modified PSO (AMPSO), and Firefly Algorithm (FA).

Keywords: microgrid, operation management, optimization, firefly algorithm (AMFA)

Procedia PDF Downloads 341

Authors: Obsi Terfasa, Bhanupriya Das, Mithilish Passawan

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The large-scale application of microbial fuel cell (MFC) technology is significantly hindered by the high cost of the commonly used proton exchange membrane, Nafion. This has led to the recent development of ceramic membranes using various clay minerals. This study evaluates the characteristics and potential use of a new ceramic membrane made from potter’s clay © mixed with different proportions (0, 5, 10 wt%) of fly ash (FA), labeled as CFA0, CFA5, CFA10, for cost-effective and sustainable MFC use. Among these, the CFA10 membrane demonstrated superior quality with a fine pore size distribution (average 0.41 μm), which supports higher water uptake and reduced oxygen diffusion. Its oxygen mass transfer coefficient was 4.13 ± 0.13 × 10⁻⁴ cm/s, about 40% lower than the control. X-ray diffraction analysis revealed that the CFA membrane is rich in quartz, which enhances proton conductance and water retention. Electrochemical kinetics studies, including cyclic voltammetry and electrochemical impedance spectroscopy (EIS), also confirmed the effectiveness of the CFA10 membrane in MFC, showing a peak current output of 15.35 mA and low ohmic resistance (78.2 Ω). The novel CFA10 ceramic membrane, incorporating coal fly ash, a waste material, shows promise for high MFC performance at a significantly reduced cost (96%), making it suitable for sustainable scaling up of the technology.

Keywords: ceramic membrane, Coulombic efficiency, electro-chemical kinetics, fly ash, proton conductivity, microbial fuel cell

Procedia PDF Downloads 36

Authors: Mohamed H. El-Sheikh, Hoda F. Zahran

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Olive tree (Olea europaea L.) is considered as a Mediterranean tree which belongs to genus Olea that may comprise about 35 species. In fact, the crop requires mild to cool winters with a chilling accumulation from November to February with average temperatures varying between two groups of accumulated chilling hours (h1) of less than 7.2 °C (C1) and other group (h2) of less than 10 °C (C2) for flower bud differentiation. This work aims at studying the impact of chilling accumulation hours on the fruit yield of olive trees in Borg El Arab City, Alexandria Governorate, Egypt as a case study. Trees were aged around 7 years in 2010 and were exposed to chilling accumulation hours of h1, which was average of 280 hours under C1, and average h2 was around 150 hours under C2 the resulted fruit yield was around 0.5 kg/tree. On the hand, trees were aged around 7 years at 2016 showed that when average of h1 was around 390 hours under C1 and average h2 was around 220 hours under C2 then fruit yield was around 10 kg/tree. Increasing of fruit yield proved chilling accumulation effect on olive trees.

Keywords: chilling accumulation, fruit yield, Olea europaea, olive

Procedia PDF Downloads 291

Authors: Okyu Kwon, Dongho Kang, Byungsik Kim, Seungkwon Jung

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We propose a technique to measure the impact of loss of traffic function in a particular area to surrounding areas. The proposed method is applied to the city of Seoul, which is the capital of South Korea, with a population of about ten million. Based on the actual road network in Seoul, we construct an abstract road network between 1kmx1km grid cells. The link weight of the abstract road network is re-adjusted considering traffic volume measured at several survey points. On the modified abstract road network, we evaluate the traffic vulnerability by calculating a network measure of betweenness centrality (BC) for every single grid cells. This study analyzes traffic impacts caused by road dysfunction due to heavy rainfall in urban areas. We could see the change of the BC value in all other grid cells by calculating the BC value once again when the specific grid cell lost its traffic function, that is, when the node disappeared on the grid-based road network. The results show that it is appropriate to use the sum of the BC variation of other cells as the influence index of each lattice cell on traffic. This research was supported by a grant (2017-MOIS31-004) from Fundamental Technology Development Program for Extreme Disaster Response funded by Korean Ministry of Interior and Safety (MOIS).

Keywords: vulnerability, road network, beweenness centrality, heavy rainfall, road impact

Procedia PDF Downloads 95

Authors: Khobaib Khobaib, Alexander Mikkelsen, Zbigniew Rozynek

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Electric fields have proven useful for inducing droplet deformation and to structure particles adsorbed at droplet interfaces. In this experimental research, direct current electric fields were applied to deform particle-covered droplets made out of silicone oil and immersed in castor oil. The viscosity of the drop and surrounding fluid were changed by external heating. We designed an experimental system in such a way that electric field-induced electrohydrodynamic (EHD) flows were asymmetric and only present on one side of the drop, i.e., the droplet adjoined a washer and adhered to one of the electrodes constituting the sample cell. The study investigated the influence of viscosity on the steady-state deformation magnitude of particle-laden droplets, droplet compression, and relaxation, as well as particle arrangements at drop interfaces. Initially, before the application of an electric field, we changed the viscosity of the fluids by heating the sample cell at different temperatures. The viscosity of the fluids was varied by changing the temperature of the fluids from 25 to 50°C. Under the application of a uniform electric field of strength 290 Vmm⁻¹, electric stress was induced at the drop interface, yielding drop deformation. In our study, we found that by lowering the fluid viscosity, the velocity of the EHD flows was increased, which also increases the deformation of the drop.

Keywords: drop deformation and relaxation, electric field, electrohydrodynamic flow, particle assembly, viscosity

Procedia PDF Downloads 265

Authors: Somdech Rungsrisawas

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The objectives of this study are to examine the relationship between the competitive advantage of small and medium enterprises (SMEs) and their overall performance. A mixed method has been applied to identify the effect of determinants toward competitive advantage. The sample is composed of SMEs in product and service businesses. The study has been tested at an organizational level with samples of SME entrepreneurs, business successors, and board of directors or management team. Quantitative analysis has been conducted through multiple regression analysis with 400 samples. The findings illustrate that each aspect of competitive advantage needs a different set of driving factors to explain either the direct or the indirect effect on firm performance. Interestingly, technological capability is a perfect mediator and interorganizational cooperation toward competitive advantage. In addition, differentiation is difficult to be perceived by customers, as well as difficult to manage; however, it is considered important to develop an SMEs product or service for firm sustainably.

Keywords: competitive advantage, firm performance, technological capability, Small and Medium Enterprise (SMEs)

Procedia PDF Downloads 297

Authors: Ádám Vass, István Bakos, Irina Borbáth, Zoltán Pászti, István Sajó, András Tompos

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Important requirements for the anode side electrocatalysts of polymer electrolyte membrane (PEM) fuel cells are CO-tolerance, stability and corrosion resistance. Carbon is still the most common material for electrocatalyst supports due to its low cost, high electrical conductivity and high surface area, which can ensure good dispersion of the Pt. However, carbon becomes degraded at higher potentials and it causes problem during application. Therefore it is important to explore alternative materials with improved stability. Molybdenum-oxide can improve the CO-tolerance of the Pt/C catalysts, but it is prone to leach in acidic electrolyte. The Mo was stabilized by isovalent substitution of molybdenum into the rutile phase titanium-dioxide lattice, achieved by a modified multistep sol-gel synthesis method optimized for preparation of Ti0.7Mo.3O2-C composite. High degree of Mo incorporation into the rutile lattice was developed. The conductivity and corrosion resistance across the anticipated potential/pH window was ensured by mixed oxide – activated carbon composite. Platinum loading was carried out using NaBH4 and ethylene glycol; platinum content was 40 wt%. The electrocatalyst was characterized by both material investigating methods (i.e. XRD, TEM, EDS, XPS techniques) and electrochemical methods (cyclic-voltammetry, COads stripping voltammetry, hydrogen oxidation reaction on rotating disc electrode). The electrochemical activity of the sample was compared to commercial 40 wt% Pt/C (Quintech) and PtRu/C (Quintech, Pt= 20 wt%, Ru= 10 wt%) references. Enhanced CO tolerance of the electrocatalyst prepared using the Ti0.7Mo.3O2-C composite material was evidenced by the appearance of a CO-oxidation related 'pre-peak' and by the pronounced shift of the maximum of the main CO oxidation peak towards less positive potential compared to Pt/C. Fuel cell polarization measurements were also carried out using Bio-Logic and Paxitech FCT-150S test device. All details on the design, preparation, characterization and testing by both electrochemical measurements and fuel cell test device of electrocatalyst supported on Ti0.7Mo.3O2-C composite material will be presented and discussed.

Keywords: anode electrocatalyst, composite material, CO-tolerance, TiMoOx

Procedia PDF Downloads 300

Authors: Annie Gedion, Johan Severinus Tati, Jacinta Caroline Peter

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Syntax error analysis studies have a significant role in English language teaching especially in the second language. This study investigates the syntax errors in written composition by 50 multilingual ESL learners in Politeknik Kota Kinabalu Sabah, Malaysia. The subjects speak their own dialect, Malay as their second language and English as their third or foreign language. Data were collected from the written discourse in the form of descriptive essays. The subjects were asked to write in the classroom within 45 minutes. 15 categories of errors were classified into a set of syntactic categories and were analysed based on the five steps of the syntactic analysis procedure. The findings of the study showed that the mother tongue interference, as well as lack of vocabulary and grammar knowledge, were the major sources of syntax errors in the learners’ written composition. Learners should be exposed to the differentiation of Malay and English grammar to avoid interference and effective learning of second language writing.

Keywords: errors analysis, syntactic analysis, English as a second language, ESL writing

Procedia PDF Downloads 283

Authors: Nkechi V. Enwuru, Bullum Nkeki, Elizabeth A. Adekoya, Olumide A. Adebesin, Rebecca F. Peters, Victoria A. Aikhomu, Mendie E. U.

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Background: Probiotics are live microbial feed supplement beneficial for host. Probiotics and their postbiotic products have been used to prevent or treat various health conditions. However, the products cell viability is often low due to harsh conditions subjected during processing, handling, storage, and gastrointestinal transit. These strongly influence probiotics’ benefits; thus, viability is essential for probiotics to produce health benefits for the host. Microencapsulation is a promising technique with considerable effects on probiotic survival. The study is aimed to formulate a microencapsulated probiotic and evaluate its viability, antimicrobial efficacy, and cytotoxic activity of its postbiotic on the MCF-7 breast cancer cell line. Method: Human and animal raw milk were sampled for lactic acid bacteria. The isolated bacteria were identified using conventional and VITEK 2 systems. The identified lactic acid bacterium was encapsulated using spray-dried and extrusion methods. The free, encapsulated, and chitosan-coated encapsulated probiotics were tested for viability in simulated-gastric intestinal (SGI) fluid and different storage conditions at refrigerated (4oC) and room (25oC) temperatures. The disintegration time and weight uniformity of the spray-dried hard gelatin capsules were tested. The antimicrobial property of free and encapsulated probiotics was tested against enteric pathogenic isolates from antiretroviral therapy (ART) treated HIV-positive patients. The postbiotic of the free cells was extracted, and its cytotoxic effect on the MCF-7 breast cancer cell line was tested through an MTT assay. Result: The Lactobacillus plantarum was isolated from animal raw milk. Zero-size hard gelatin L. plantarum capsules with granules within a size range of 0.71–1.00 mm diameter was formulated. The disintegration time ranges from 2.14±0.045 to 2.91±0.293 minutes, while the average weight is 502.1mg. Simulated gastric solution significantly affected viability of both free and microcapsules. However, the encapsulated cells were more protected and viable due to impermeability in the microcapsules. Furthermore, the viability of free cells stored at 4oC and 25oC were less than 4 log CFU/g and 6 log CFU/g respectively after 12 weeks. However, the microcapsules stored at 4oC achieved the highest viability among the free and microcapsules stored at 25oC and the free cells stored at 4oC. Encapsulated cells were released in the simulated gastric fluid, viable and effective against the enteric pathogens tested. However, chitosan-coated calcium alginate encapsulated probiotics significantly inhibited Shigella flexneri, Candida albicans, and Escherichia coli. The Postbiotic Metabolites (PM) of L. plantarum produced a cytotoxic effect on the MCF-7 breast cancer cell line. The postbiotic showed significant cytotoxic activity similar to 5FU, a standard antineoplastic agent. The inhibition concentration of 50% growth (IC50) of postbiotic metabolite K3 is low and consistent with the IC50 of the positive control (Cisplatin). Conclusions: Lactobacillus plantarum postbiotic exhibited a cytotoxic effect on the MCF-7 breast cancer cell line and could be used as combined adjuvant therapy in breast cancer management. The microencapsulation technique protects the probiotics, improving their viability and delivery to the gastrointestinal tract. Chitosan enhances antibacterial efficacy; thus, chitosan-coated microencapsulated L. plantarum probiotics could be more effective and used as a combined therapy in HIV management of opportunistic enteric infection.

Keywords: probiotics, encapsulation, gastrointestinal conditions, antimicrobial effect, postbiotic, cytotoxicity effect

Procedia PDF Downloads 123

Authors: Omar Hussein El Ayadi, EmadY. El-Mansouri, Mohamed B. Dozan

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Production oil process require specific details i.e. oil composition. Generally, types of oil or differentiation between reservoir fluids depend specifically on composition. The main purpose of this study is to correlate and predict the Libyan oil (reservoir fluid and residual) composition utilizing tri-angle-coordinate plots discovered and tasked with Excel. The reservoir fluid data (61 old + 47 new), the residual oil data (33 new) collected from most of Libyan reservoirs were correlated with each others. Moreover, find a relation between stock tank molecular weight and stock tank oil gravity (oAPI), the molecular weight oh (C7+) versus residual oil gravity (oAPI). The average value of every oil composition was estimated including non-hydrocarbon (H2S, CO2, and N2). Nevertheless, the isomers (i-…) and normal (n-…) structure of (C4) and (C5) were also obtained. The summary of the conclusion is; utilizing excel Microsoft office to draw triangle coordinates to find two unknown component if only one is known. However, it is recommended to use the obtained oil composition plots and equations for any oil composition dependents i.e. optimum separator pressure.

Keywords: PVT, phase behavior, petroleum, chemical engineering

Procedia PDF Downloads 514

Authors: Hemant Kumar, R. N. K. Sharma, A. P. Krishna

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The study has been carried out on the Saranda in Jharkhand and a part of Odisha state. Geospatial data of Hyperion, a remote sensing satellite, have been used. This study has used a wide variety of patterns related to image processing to enhance and extract the mining class of Fe and Mn ores.Landsat-8, OLI sensor data have also been used to correctly explore related minerals. In this way, various processes have been applied to increase the mineralogy class and comparative evaluation with related frequency done. The Hyperion dataset for hyperspectral remote sensing has been specifically verified as an effective tool for mineral or rock information extraction within the band range of shortwave infrared used. The abundant spatial and spectral information contained in hyperspectral images enables the differentiation of different objects of any object into targeted applications for exploration such as exploration detection, mining.

Keywords: Hyperion, hyperspectral, sensor, Landsat-8

Procedia PDF Downloads 123

Authors: Yanni Chang, Dezhi Dai, Albert Y. Tong

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Piecewise linear interface calculation (PLIC) schemes are widely used in the volume-of-fluid (VOF) method to capture interfaces in numerical simulations of multiphase flows. Dynamic overset meshes can be especially useful in applications involving component motions and complex geometric shapes. In the present study, the VOF value of an acceptor cell is evaluated in a geometric way that transfers the fraction field between the meshes precisely with reconstructed interfaces from the corresponding donor elements. The acceptor cell value is evaluated by using a weighted average of its donors for most of the overset interpolation schemes for continuous flow variables. The weighting factors are obtained by different algebraic methods. Unlike the continuous flow variables, the VOF equation is a step function near the interfaces, which ranges from zero to unity rapidly. A geometric interpolation scheme of the VOF field in overset meshes for the PLIC-VOF method has been proposed in the paper. It has been tested successfully in quadrilateral/hexahedral overset meshes by employing several VOF advection tests with imposed solenoidal velocity fields. The proposed algorithm has been shown to yield higher accuracy in mass conservation and interface reconstruction compared with three other algebraic ones.

Keywords: interpolation scheme, multiphase flows, overset meshes, PLIC-VOF method

Procedia PDF Downloads 176

Authors: F. Kashanian, M. M. Masoudi, A. Akbari, A. Shamloo, M. R. Zand, S. S. Salehi

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Nano-sized materials present new opportunities in biology and medicine and they are used as biomedical tools for investigation, separation of molecules and cells. To achieve more effective cancer therapy, it is essential to select cancer cells exactly. This research suggests that using the antibody-functionalized nontoxic Arginine-doped magnetic nanoparticles (A-MNPs), has been prosperous in detection, capture, and magnetic separation of circulating tumor cells (CTCs) in tumor tissue. In this study, A-MNPs were synthesized via a simple precipitation reaction and directly immobilized Ep-CAM EBA-1 antibodies over superparamagnetic A-MNPs for Mucin BCA-225 in breast cancer cell. The samples were characterized by vibrating sample magnetometer (VSM), FT-IR spectroscopy, Tunneling Electron Microscopy (TEM) and Scanning Electron Microscopy (SEM). These antibody-functionalized nontoxic A-MNPs were used to capture breast cancer cell. Through employing a strong permanent magnet, the magnetic separation was achieved within a few seconds. Antibody-Conjugated nontoxic Arginine-doped Fe₃O₄ nanoparticles have the potential for the future study to capture CTCs which are released from tumor tissue and for drug delivery, and these results demonstrate that the antibody-conjugated A-MNPs can be used in magnetic hyperthermia techniques for cancer treatment.

Keywords: tumor tissue, antibody, magnetic nanoparticle, CTCs capturing

Procedia PDF Downloads 360

Authors: Lynn Lehmann, Dinorah Leyva, Ana Lazic, Stefan Duhr, Philipp Baaske

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Characterization of biomolecular interactions, such as protein-protein, protein-nucleic acid or protein-small molecule, provides critical insights into cellular processes and is essential for the development of drug diagnostics and therapeutics. Here we present a novel, label-free, and tether-free technology to analyze picomolar to millimolar affinities of biomolecular interactions by Microscale Thermophoresis (MST). The entropy of the hydration shell surrounding molecules determines thermophoretic movement. MST exploits this principle by measuring interactions using optically generated temperature gradients. MST detects changes in the size, charge and hydration shell of molecules and measures biomolecule interactions under close-to-native conditions: immobilization-free and in bioliquids of choice, including cell lysates and blood serum. Thus, MST measures interactions under close-to-native conditions, and without laborious sample purification. We demonstrate how MST determines the picomolar affinities of antibody::antigen interactions, and protein::protein interactions measured from directly from cell lysates. MST assays are highly adaptable to fit to the diverse requirements of different and complex biomolecules. NanoTemper´s unique technology is ideal for studies requiring flexibility and sensitivity at the experimental scale, making MST suitable for basic research investigations and pharmaceutical applications.

Keywords: biochemistry, biophysics, molecular interactions, quantitative techniques

Procedia PDF Downloads 523

Authors: Matan Cohen, Maxim Shoshany

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Climatic, topographic and geological diversity, together with frequent disturbance and recovery cycles, produce highly complex spatial patterns of trees, shrubs, dwarf shrubs and bare ground patches. Assessment of spatial and temporal variations of these life-forms patterns under climate change is of high ecological priority. Here we report on one of the first attempts to discriminate between images of three Mediterranean life-forms patterns at three densities. The development of an extensive database of orthophoto images representing these 9 pattern categories was instrumental for training and testing pre-trained and newly-trained DL models utilizing DenseNet architecture. Both models demonstrated the advantages of using Deep Learning approaches over existing spectral and spatial (pattern or texture) algorithmic methods in differentiation 9 life-form spatial mixtures categories.

Keywords: texture classification, deep learning, desert fringe ecosystems, climate change

Procedia PDF Downloads 88

Authors: Drago Bratkovic, Curtis Gravance, David Ketteridge, Ravi Krishnan, Michael Imperiale

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The mucopolysaccharidoses (MPSs) are a group of lysosomal storage diseases that have a common defect in the catabolism of glycosaminoglycans (GAGs). MPS I is the most common of the MPS diseases. Manifestations of MPS I include coarsening of facial features, corneal clouding, developmental delay, short stature, skeletal manifestations, hearing loss, cardiac valve disease, hepatosplenomegaly, and umbilical and inguinal hernias. Treatments for MPS I restore or activate the missing or deficient enzyme in the case of enzyme replacement therapy (ERT) and haematopoietic stem cell transplantation (HSCT). Pentosan polysulfate sodium (PPS) is a potential treatment to improve bone and joint manifestations of MPS I. The mechanisms of action of PPS that are relevant to the treatment of MPS I are the ability to: (i) Reduce systemic and accumulated GAG, (ii) Reduce inflammatory effects via the inhibition of NF-kB, resulting in the reduction in pro-inflammatory mediators. (iii) Reduce the expression of the pain mediator nerve growth factor in osteocytes from degenerating joints. (iv) Inhibit the cartilage degrading enzymes related to joint dysfunction in MPS I. PPS is being evaluated as an adjunctive therapy to ERT and/or HSCT in an open-label, single-centre, phase 2 study. Patients are ≥ 5 years of age with a diagnosis of MPS I and previously received HSCT and/or ERT. Three white, female, patients with MPS I-Hurler, ages 14, 15, and 19 years, and one, white male patient aged 15 years are enrolled. All were diagnosed at ≤2 years of age. All patients received HSCT ≤ 6 months after diagnosis. Two of the patients were treated with ERT prior to HSCT, and 1 patient received ERT commencing 3 months prior to HSCT. Two patients received 0.75mg/kg and 2 patients received 1.5mg/kg of PPS. PPS was well tolerated at doses of 0.75 and 1.5 mg/kg to 47 weeks of continuous dosing. Of the 19 adverse events (AEs), 2 were related to PPS. One AE was moderate (pre-syncope) and 1 was mild (injection site bruising), experienced in the same patient. All AEs were reported as mild or moderate. There have been no SAEs. One subject experienced a COVID-19 infection and PPS was interrupted. The MPS I signature GAG fragments, sulfated disaccharide and UA-HNAc S, tended to decrease in 3 patients from baseline through Week 25. Week 25 GAG data are pending for the 4th patient. Overall, most biomarkers (inflammatory, cartilage degeneration, and bone turnover) evaluated in the 3 patients with 25-week assessments have indicated either no change or a reduction in levels compared to baseline. In 3 patients, there was a trend toward improvement in the 2MWT from baseline to Week 48 with > 100% increase in 1 patient (01-201). In the 3 patients that had Week 48 assessments, patients and proxies reported improvement in PGIC, including “worthwhile difference” (n=1), or “made all the difference” (n=2).

Keywords: MPS I, pentosan polysulfate sodium, clinical study, 2MWT, QoL

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Authors: E. Nakamachi, S. Tanaka, K. Yamamoto, Y. Morita

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In this study, we developed a three-dimensional (3D) direct current electric field (DCEF) stimulation bio-reactor for axonal outgrowth enhancement to generate the neural network of the central nervous system (CNS). By using our newly developed 3D DCEF stimulation bio-reactor, we cultured the rat pheochromocytoma cells (PC12) and investigated the effects on the axonal extension enhancement and network generation. Firstly, we designed and fabricated a 3D bio-reactor, which can load DCEF stimulation on PC12 cells embedded in the collagen gel as extracellular environment. The connection between the electrolyte and the medium using salt bridges for DCEF stimulation was introduced to avoid the cell death by the toxicity of metal ion. The distance between the salt bridges was adopted as the design variable to optimize a structure for uniform DCEF stimulation, where the finite element (FE) analyses results were used. Uniform DCEF strength and electric flux vector direction in the PC12 cells embedded in collagen gel were examined through measurements of the fabricated 3D bio-reactor chamber. Measurement results of DCEF strength in the bio-reactor showed a good agreement with FE results. In addition, the perfusion system was attached to maintain pH 7.2 ~ 7.6 of the medium because pH change was caused by DCEF stimulation loading. Secondly, we disseminated PC12 cells in collagen gel and carried out 3D culture. Finally, we measured the morphology of PC12 cell bodies and neurites by the multiphoton excitation fluorescence microscope (MPM). The effectiveness of DCEF stimulation to enhance the axonal outgrowth and the neural network generation was investigated. We confirmed that both an increase of mean axonal length and axogenesis rate of PC12, which have been exposed 5 mV/mm for 6 hours a day for 4 days in the bioreactor. We found following conclusions in our study. 1) Design and fabrication of DCEF stimulation bio-reactor capable of 3D culture nerve cell were completed. A uniform electric field strength of average value of 17 mV/mm within the 1.2% error range was confirmed by using FE analyses, after the structure determination through the optimization process. In addition, we attached a perfusion system capable of suppressing the pH change of the culture solution due to DCEF stimulation loading. 2) Evaluation of DCEF stimulation effects on PC12 cell activity was executed. The 3D culture of PC 12 was carried out adopting the embedding culture method using collagen gel as a scaffold for four days under the condition of 5.0 mV/mm and 10mV/mm. There was a significant effect on the enhancement of axonal extension, as 11.3% increase in an average length, and the increase of axogenesis rate. On the other hand, no effects on the orientation of axon against the DCEF flux direction was observed. Further, the network generation was enhanced to connect longer distance between the target neighbor cells by DCEF stimulation.

Keywords: PC12, DCEF stimulation, 3D bio-reactor, axonal extension, neural network generation

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Authors: Chang-Hui Shen, Paulina Konarzewska

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Cell homeostasis is regulated by vacuolar activity and it has been shown that lipid composition of the vacuole plays an important role in vacuolar function. The major phosphoinositide species present in the vacuolar membrane include phosphatidylinositol 3,5-biphosphate (PI(3,5)P₂) which is generated from PI(3)P controlled by Fab1p. Deletion of FAB1 gene reduce the synthesis of PI(3,5)P₂ and thus result in enlarged or fragmented vacuoles, with neutral vacuolar pH due to reduced vacuolar H⁺-ATPase activity. These mutants also exhibited poor growth at high extracellular pH and in the presence of CaCl₂. Conversely, VPS34 regulates the synthesis of PI(3)P from phosphatidylinositol (PI), and the lack of Vps34p results in the reduction of vacuolar activity. Although the cellular observations are clear, it is still unknown about the molecular mechanism between the phospholipid biosynthesis pathway and vacuolar activity. Since both VPS34 and FAB1 are important in vacuolar activity, we hypothesize that the molecular mechanism of vacuolar function might be regulated by the transcriptional regulators of phospholipid biosynthesis. In this study, we study the role of the major phospholipid biosynthesis transcription factor, INO2, in the regulation of vacuolar activity. We first performed qRT-PCR to examine the effect of Ino2p on the expression of VPS34 and FAB1. Our results showed that VPS34 was upregulated in the presence of inositol for both WT and ino2Δ cells. However, FAB1 was only upregulated significantly in ino2Δ cells. This indicated that Ino2p might be the negative regulator for FAB1 expression. Next, growth sensitivity experiment showed that WT, vma3Δ, and ino2Δ grew well in growth medium buffered to pH 5.5 containing 10 mM CaCl₂. As cells were switched to growth medium buffered to pH 7 containing CaCl₂ WT, ino2Δ and opi1Δ showed growth reduction, whereas vma3Δ was completely nonviable. As the concentration of CaCl₂ was increased to 60 mM, ino2Δ cells showed moderate growth reduction compared to WT. This result suggests that ino2Δ cells have better vacuolar activity. Microscopic analysis and vacuolar acidification were employed to further elucidate the importance of INO2 in vacuolar homeostasis. Analysis of vacuolar morphology indicated that WT and vma3Δ cells displayed vacuoles that occupied a small area of the cell when grown in media buffered to pH 5.5. Whereas, ino2Δ displayed fragmented vacuoles. On the other hand, all strains grown in media buffered to pH 7, exhibited enlarged vacuoles that occupied most of the cell’s surface. This indicated that the presence of INO2 may play negative effect in vacuolar morphology when cells are grown in media buffered to pH 5.5. Furthermore, vacuolar acidification assay showed that only vma3Δ cells displayed notably less acidic vacuoles as cells were grown in media buffered to pH 5.5 and pH 7. Whereas, ino2Δ cells displayed more acidic pH compared to WT at pH7. Taken together, our results demonstrated the molecular mechanism of the vacuolar activity regulated by the phospholipid biosynthesis transcription factors Ino2p. Ino2p negatively regulates vacuolar activity through the expression of FAB1.

Keywords: vacuole, phospholipid, homeostasis, Ino2p, FAB1

Procedia PDF Downloads 127

Authors: Ayda Youssef, Tarek Rafaat, Iman zaky

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Purpose: Langerhans’ cell histiocytosis (LCH) is not uncommon pathology that implies aberrant proliferation of a specific dendritic (Langerhans) cell. These atypical but mature cells of monoclonal origin can infiltrate many sites of the body and may occur as localized lesions or as widespread systemic disease. Liver is one of the uncommon sites of affection. The twofold objective of this study is to illustrate the radiological presentation of this disease, and to compare these results with previously reported series. Methods and Materials: Between 2007 and 2012, 150 patients with biopsy-proven LCH were treated in our hospital, a paediatric cancer tertiary care center. A retrospective review of radiographic images and reports was performed. There were 33 patients with liver affection are stratified. All patients underwent imaging studies, mostly US and CT. A chart review was performed to obtain demographic, clinical and radiological data. They were analyzed and compared to other published series. Results: Retrospective assessment of 150 patients with LCH was performed, among them 33 patients were identified who had liver involvement. All these patients developed multisystemic disease; They were 12 females and 21 males with (n= 32), seven of them had marked hepatomegaly. Diffuse hypodense liver parenchyma was encountered in five cases, the periportal location has a certain predilection in cases of focal affection where three cases has a hypodense periportal soft tissue sheets, one of them associated with dilated biliary radicals, only one case has multiple focal lesions unrelated to portal tracts. On follow up of the patients, two cases show abnormal morphology of liver with bossy outline. Conclusion: LCH is a not infrequent disease. A high-index suspicion should be raised in the context of diagnosis of liver affection. A biopsy is recommended in the presence of radiological suspicion. Chemotherapy is the preferred therapeutic modality. Liver histiocytosis are not disease specific features but should be interpreted in conjunction with the clinical history and the results of biopsy. Clinical Relevance/Application: Radiologist should be aware of different patterns of hepatobiliary histiocytosis, Thus early diagnosis and proper management of patient can be conducted.

Keywords: langerhans’ cell histiocytosis, liver, medical and health sciences, radiology

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Authors: Anna Cameron, Chunxia Zhao, Haofei Wang, Yun Liu, Guang Ze Yang

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Since the first publication in 1775, cancer research has built a comprehensive understanding of how cellular components of the tumor niche promote disease development. However, only within the last decade has research begun to establish the impact of non-cellular components of the niche, particularly the extracellular matrix (ECM). The ECM, a three-dimensional scaffold that sustains the tumor microenvironment, plays a crucial role in disease progression. Cancer cells actively deregulate and remodel the ECM to establish a tumor-promoting environment. Recent work has highlighted the need to further our understanding of the complexity of this cancer-ECM relationship. In vitro models use hydrogels to mimic the ECM, as hydrogel matrices offer biological compatibility and stability needed for long term cell culture. However, natural hydrogels are being used in these models verbatim, without tuning their biophysical characteristics to achieve pathophysiological relevance, thus limiting their broad use within cancer research. The biophysical attributes of these gels dictate cancer cell proliferation, invasion, metastasis, and therapeutic response. Evaluating the three most widely used natural hydrogels, Matrigel, collagen, and agarose gel, the permeability, stiffness, and pore-size of each gel were measured and compared to the in vivo environment. The pore size of all three gels fell between 0.5-6 µm, which coincides with the 0.1-5 µm in vivo pore size found in the literature. However, the stiffness for hydrogels able to support cell culture ranged between 0.05 and 0.3 kPa, which falls outside the range of 0.3-20,000 kPa reported in the literature for an in vivo ECM. Permeability was ~100x greater than in vivo measurements, due in large part to the lack of cellular components which impede permeation. Though, these measurements prove important when assessing therapeutic particle delivery, as the ECM permeability decreased with increasing particle size, with 100 nm particles exhibiting a fifth of the permeability of 10 nm particles. This work explores ways of adjusting the biophysical characteristics of hydrogels by changing protein concentration and the trade-off, which occurs due to the interdependence of these factors. The global aim of this work is to produce a more pathophysiologically relevant model for each tumor type.

Keywords: cancer, extracellular matrix, hydrogel, microfluidic

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Authors: Shanna Swart, Caryn Fenner, Athanasios Kotsiopoulos, Susan Harrison

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Linear alkanes are produced as by-products from the increasing use of gas-to-liquid fuel technologies for synthetic fuel production and offer great potential for value addition. Their current use as low-value fuels and solvents do not maximize this potential. Therefore, attention has been drawn towards direct activation of these aliphatic alkanes to more useful products such as alcohols, aldehydes, carboxylic acids and derivatives. Cytochrome P450 monooxygenases (P450s) can be used for activation of these aliphatic alkanes using whole-cells or cell-free systems. Some limitations of whole-cell systems include reduced mass transfer, stability and possible side reactions. Since the P450 systems are little studied as cell-free systems, they form the focus of this study. Challenges of a cell-free system include co-factor regeneration, substrate availability and enzyme stability. Enzyme immobilization offers a positive outlook on this dilemma, as it may enhance stability of the enzyme. In the present study, 2 different P450s (CYP153A6 and CYP102A1) as well as the relevant accessory enzymes required for electron transfer (ferredoxin and ferredoxin reductase) and co-factor regeneration (glucose dehydrogenase) have been expressed in E. coli and purified by metal affinity chromatography. Glucose dehydrogenase (GDH), was used as a model enzyme to assess the potential of various enzyme immobilization strategies including; surface attachment on MagReSyn® microspheres with various functionalities and on electrospun nanofibers, using self-assembly based methods forming Cross Linked Enzymes (CLE), Cross Linked Enzyme Aggregates (CLEAs) and spherezymes as well as in a sol gel. The nanofibers were synthesized by electrospinning, which required the building of an electrospinning machine. The nanofiber morphology has been analyzed by SEM and binding will be further verified by FT-IR. Covalent attachment based methods showed limitations where only ferredoxin reductase and GDH retained activity after immobilization which were largely attributed to insufficient electron transfer and inactivation caused by the crosslinkers (60% and 90% relative activity loss for the free enzyme when using 0.5% glutaraldehyde and glutaraldehyde/ethylenediamine (1:1 v/v), respectively). So far, initial experiments with GDH have shown the most potential when immobilized via their His-tag onto the surface of MagReSyn® microspheres functionalized with Ni-NTA. It was found that Crude GDH could be simultaneously purified and immobilized with sufficient activity retention. Immobilized pure and crude GDH could be recycled 9 and 10 times, respectively, with approximately 10% activity remaining. The immobilized GDH was also more stable than the free enzyme after storage for 14 days at 4˚C. This immobilization strategy will also be applied to the P450s and optimized with regards to enzyme loading and immobilization time, as well as characterized and compared with the free enzymes. It is anticipated that the proposed immobilization set-up will offer enhanced enzyme stability (as well as reusability and easy recovery), minimal mass transfer limitation, with continuous co-factor regeneration and minimal enzyme leaching. All of which provide a positive outlook on this robust multi-enzyme system for efficient activation of linear alkanes as well as the potential for immobilization of various multiple enzymes, including multimeric enzymes for different bio-catalytic applications beyond alkane activation.

Keywords: alkane activation, cytochrome P450 monooxygenase, enzyme catalysis, enzyme immobilization

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Authors: Mengfei Peng, Serajus Salaheen, Debabrata Biswas

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Lactobacillus casei found in the human intestine and mouth is commonly applied for dairy production. Recently, it was found that some byproducts produced by Lactobacillus exhibited antimicrobial activities against multiple bacteria. Meanwhile, introduction of prebiotic-like foods (e.g. cocoa) or probiotics or both of them as food supplements in human diets as well as in farm animal feeds is believed to be an effective ways in control/reduce the colonization of foodborne bacterial pathogens infection in the gut environment. We hypothesized that cocoa may stimulate the production antimicrobial components of Lactobacillus casei and may potentially inhibit/reduce the colonization and infection of foodborne bacterial pathogens in the gut. Mixed culture of L. casei (LC) with enterohemorrhagic E. coli EDL933 (EHEC), Salmonella Typhimurium LT2 (ST), or Listeria monocytogenes LM2 (LM) showed that LC could competitively exclude (100%) them within 72 h. Further, investigation of cell-free culture supernatant (CFCS) revealed that the antimicrobial effects of LC came from CFCS. CFCS of LC eliminated (100%) EHEC, ST, and LM within 72 h, and 2 h CFCS treatment increased the hydrophobicity of EHEC (5.10 folds), ST (8.48 folds), and LM (2.03 folds). In addition, LC cells exhibited more inhibitive effects than CFCS on cell adhesive and invasive activities of EHEC (52.14% & 90.45%), ST (66.89% & 93.83%), and LM (61.10% & 83.40%). Two clusters of poly-peptides in CFCS were identified by SDS-PAGE, the molecular weights of which are ≈5 KD and 40-45 KD. LC CFCS with overnight growth in the presence of 3% strengthened all of the antimicrobial activities (growth inhibition, outer membrane disruption, and cell infective ability reduction). Liquid chromatography/Mass spectrometry analysis detected 5 unique components in class of flavonoids in LC CFCS with overnight 3% cocoa supplement. Furthermore, qPCR results showed that CFCSs up-regulated the expression level of genes responsible for flagellin synthesis and motility, but down-regulated genes for specific binding and invasion-associated proteins synthesis. The stimulatory effects of cocoa in producing bioactive components of probiotics may aid prevention of foodborne illness caused by major foodborne enteric bacterial pathogens.

Keywords: foodborne pathogens, probiotics, prebiotics, pathogen exclusion

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Authors: Xin Chen, Xinyong Li, Qidong Zhao, Dong Wang

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A new coupling system was constructed by combining photo-electrochemical cell with electro-fenton cell (PEC-EF). The electrode material in this system was derived from MnyFe₁₋yCo Prussian-Blue-Analog (PBA). Mn₀.₄Fe₀.₆Co₀.₆₇-N@C spin-coated on carbon paper behaved as the gas diffusion cathode and Mn₀.₄Fe₀.₆Co₀.₆₇O₂.₂ spin-coated on fluorine-tin oxide glass (FTO) as anode. The two separated cells could degrade Sulfamethoxazole (SMX) simultaneously and some coupling mechanisms by PEC and EF enhancing the degradation efficiency were investigated. The continuous on-site generation of H₂O₂ at cathode through an oxygen reduction reaction (ORR) was realized over rotating ring-disk electrode (RRDE). The electron transfer number (n) of the ORR with Mn₀.₄Fe₀.₆Co₀.₆₇-N@C was 2.5 in the selected potential and pH range. The photo-electrochemical properties of Mn₀.₄Fe₀.₆Co₀.₆₇O₂.₂ were systematically studied, which displayed good response towards visible light. The photoinduced electrons at anode can transfer to cathode for further use. Efficient photo-electro-catalytic performance was observed in degrading SMX. Almost 100% SMX removal was achieved in 120 min. This work not only provided a highly effective technique for antibiotic treatment but also revealed the synergic effect between PEC and EF.

Keywords: electro-fenton, photo-electrochemical, synergic effect, sulfamethoxazole

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Authors: Ali Mirtaghavi, Andy Baldwin, Rajendarn Muthuraj, Jack Luo

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Recent advances in biomaterials have led to utilizing biopolymers to develop 3D scaffolds in tissue regeneration. One of the major challenges of designing biomaterials for 3D scaffolds is to mimic the building blocks similar to the extracellular matrix (ECM) of the native tissues. Biopolymer based aerogels obtained by freeze-drying have shown to provide structural similarities to the ECM owing to their 3D format and a highly porous structure with interconnected pores, similar to the ECM. Gelatin (GEL) is known to be a promising biomaterial with inherent regenerative characteristics owing to its chemical similarities to the ECM in native tissue, biocompatibility abundance, cost-effectiveness and accessible functional groups, which makes it facile for chemical modifications with other biomaterials to form biocomposites. Despite such advantages, gelatin offers poor mechanical properties, sensitive enzymatic degradation and high viscosity at room temperature which limits its application and encourages its use to develop biocomposites. Hydrophilic biomass-based cellulose nanofibrous (CNF) has been explored to use as suspension for biocomposite aerogels for the development of 3D porous structures with excellent mechanical properties, biocompatibility and slow enzymatic degradation. In this work, CNF biocomposite aerogels with various ratios of CNF:GEL) (90:10, 70:30 and 50:50) were prepared by freeze-drying technique, and their properties were investigated in terms of physicochemical, mechanical and biological characteristics. Epichlorohydrin (EPH) was used to investigate the effect of chemical crosslinking on the molecular interaction of CNF: GEL, and its effects on physicochemical, mechanical and biological properties of the biocomposite aerogels. Ultimately, chemical crosslinking helped to improve the mechanical resilience of the resulting aerogels. Amongst all the CNF-GEL composites, the crosslinked CNF: GEL (70:30) biocomposite was found to be favourable for cell attachment and viability. It possessed highly porous structure (porosity of ~93%) with pore sizes ranging from 16-110 µm, adequate mechanical properties (compression modulus of ~47 kPa) and optimal biocompatibility both in-vitro and in-vivo, as well as controlled enzymatic biodegradation, high water penetration, which could be considered a suitable option for wound healing application. In-vivo experiments showed improvement on inflammation and foreign giant body cell reaction for the crosslinked CNF: GEL (70:30) compared to the other samples. This could be due to the superior interaction of CNF with gelatin through chemical crosslinking, resulting in more optimal in-vivo improvement. In-vitro cell culture investigation on human dermal fibroblasts showed satisfactory 3D cell attachment over time. Overall, it has been observed that the developed CNF: GEL aerogel can be considered as a potential scaffold for soft tissue regeneration application.

Keywords: 3D scaffolds, aerogels, Biocomposites , tissue engineering

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Authors: Mrinal Gupta, Mohammad Rumman, Babita Singh Abbas Ali Mahdi, Shivani Pandey

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Introduction: Berberine (BBR), a bioactive compound isolated from Coptidis Rhizoma, possesses diverse pharmacological activities, including anti-bacterial, anti-inflammatory, antitumor, hypolipidemic, and anti-diabetic. However, its role as an anti-diabetic agent in animal models of dexamethasone (Dex)-induced diabetes remains unknown. Studies have shown that natural compounds, including aloe, caper, cinnamon, cocoa, green and black tea, and turmeric, can be used for treating Type 2 diabetes mellitus (DM). Compared to conventional drugs, natural compounds have fewer side effects and are easily available. Herein, we studied the anti-diabetic effects of BBR in a mice model of Dex-induced diabetes. Methods: HepG2 cell line was used for glucose release and glycogen synthesis studies. Cell proliferation was measured by methylthiotetrazole (MTT) assay. For animal studies, mice were treated with Dex (2 mg/kg, i.m.) for 30 days and the effect of BBR at the doses 100, 200, and 500 mg/kg (p.o.) was analyzed. Glucose, insulin, and pyruvate tests were performed to evaluate the development of the diabetic model. An echo MRI was performed to assess the fat mass. Further, to elucidate the mechanism of action of BBR, mRNA expression of genes regulating gluconeogenesis, glucose uptake, and glycolysis were analyzed. Results: In vitro BBR had no impact on cell viability up to a concentration of 50μM. Moreover, BBR suppressed the hepatic glucose release and improved glucose tolerance in HepG2 cells. In vivo, BBR improved glucose homeostasis in diabetic mice, as evidenced by enhanced glucose clearance, increased glycolysis, elevated glucose uptake, and decreased gluconeogenesis. Further, Dex treatment increased the total fat mass in mice, which was ameliorated by BBR treatment. Conclusion: BBR improves glucose tolerance by increasing glucose clearance, inhibiting hepatic glucose release, and decreasing obesity. Thus, BBR may become a potential therapeutic agent for treating glucocorticoid-induced diabetes and obesity in the future.

Keywords: glucocorticoid, hyperglycemia, berberine, HepG2 cells, insulin resistance, glucose

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Authors: Hawon Lee, Young-Pil Kim

Abstract:

Visualizing matrix metalloproteinases (MMPs) activity is necessary for understanding cancer metastasis because they are implicated in cell migration and invasion by degrading the extracellular matrix (ECM). While much effort has been made to sense the MMP activity, but extracellularly long-term monitoring of MMP activity still remains challenging. Here, we report a collagen-bound fluorescent bioprobe for the detection of MMP-2 activity in the extracellular environment. This bioprobe consists of ECM-immobilized part (including collagen-bound protein) and MMP-sensing part (including peptide substrate linked with fluorescence resonance energy transfer (FRET) coupler between donor green fluorescent protein (GFP) and acceptor TAMRA dye), which was constructed through intein-mediated self-splicing conjugation. Upon being immobilized on the collagen-coated surface, this bioprobe enabled efficient long-lasting observation of MMP-2 activity in the cultured cells without affecting cell growth and viability. As a result, the FRET ratio (acceptor/donor) decreased as the MMP2 activity increased in cultured cancer cells. Furthermore, unlike wild-type MMP-2, mutated MMP-2 expression (Y580A in the hemopexin region) gave rise to lowering the secretion of MMP-2 in HeLa. Conclusively, our method is anticipated to find applications for tracing and visualizing enzyme activity.

Keywords: collagen, ECM, FRET, MMP

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Authors: Cody Kirby, Kaustav Misra, Arundhati Bagchi Misra, Sharon P. Cox

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This paper focuses on the relationship that dual-enrollment programs have on academic performance and retention. Both performance and retention are significant issues in higher education. The first, performance, is a goal of higher education, having an impact on students’ lives. The second, retention, is key to the viability of any college or university. This paper uses survey research methodology to examine factors that lead to positive student academic performance, which leads to retention, specifically in dual-enrollment programs. The data show several characteristics that lead to a positive impact on GPA. These include the following; age, Caucasian race, full-time status, students in STEM programs, and finally dual enrollment participation.

Keywords: dual enrollment, early college, retention, undergraduate education

Procedia PDF Downloads 150

Authors: Ozlem Oral, Emre Taskin, Aysel Yuce, Serap Dokmeci, Devrim Gozuacik

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Autophagy is an evolutionary conserved lysosome-dependent catabolic pathway, responsible for the degradation of long-lived proteins, abnormal aggregates and damaged organelles which cannot be degraded by the ubiquitin-proteasome system. Lysosomes degrade the substrates through the activity of lysosomal hydrolases and lysosomal membrane-bound proteins. Mutations in the coding region of these proteins cause malfunctional lysosomes, which contributes to the pathogenesis of lysosomal storage diseases. Gaucher disease is a lysosomal storage disease resulting from the mutation of a lysosomal membrane-associated glycoprotein called glucocerebrosidase and its cofactor saposin C. The disease leads to intracellular accumulation of glucosylceramide and other glycolipids. Because of the essential role of lysosomes in autophagic degradation, Gaucher disease may directly be linked to this pathway. In this study, we investigated the expression of autophagy and/or lysosome-related genes and proteins in fibroblast cells isolated from patients with different mutations. We carried out confocal microscopy analysis and examined autophagic flux by utilizing the differential pH sensitivities of RFP and GFP in mRFP-GFP-LC3 probe. We also evaluated lysosomal pH by active lysosome staining and lysosomal enzyme activity. Beside lysosomes, we also performed proteasomal activity and cell death analysis in patient samples. Our data showed significant attenuation in the expression of key autophagy-related genes and accumulation of their proteins in mutant cells. We found decreased the ability of autophagosomes to fuse with lysosomes, associated with elevated lysosomal pH and reduced lysosomal enzyme activity. Proteasomal degradation and cell death analysis showed reduced proteolytic activity of the proteasome, which consequently leads to increased susceptibility to cell death. Our data indicate that the major degradation pathways are affected by multifunctional lysosomes in mutant patient cells and may underlie in the mechanism of clinical severity of Gaucher patients. (This project is supported by TUBITAK-3501-National Young Researchers Career Development Program, Project No: 112T130).

Keywords: autophagy, Gaucher's disease, glucocerebrosidase, mutant fibroblasts

Procedia PDF Downloads 324